Probing biosynthesis of plant polyketides with synthetic N-acetylcysteamine thioesters

Cloning and Functional Analysis of a Novel Aldo-Keto Reductase from Aloe arborescens

“…Thus, co-incubation with plant polyketide synthases (A. arborescence pentaketide chromone synthase, 18) octaketide synthase, 19) or Scutellaria baicalensis CHS 17) ) did not afford any detectable reduced products, which was confirmed by HPLC and LC-ESI-MS analysis. Instead, the recombinant enzyme efficiently reduced a wide variety of carbonyl compounds (Fig.…”

“…Interestingly, the enzyme also reduced triacetic acid lactone, and cinnamoyldiketide NAC which is the substrate for PKR. 17) To further characterize A. arborescens AKR4C12, a homology model was constructed based on the crystal structure of H. vulgare aldose reductase (4C1) (43.5% amino acid sequence identity) bound with NADPH (PDB code: 2BGS). 15) As in the case of other AKR superfamily enzymes, 2) the homology model predicted that A. arborescens AKR4C12 adopts an (a/b) 8 -barrel motif, with each of eight parallel bstrands alternating with an a-helix running anti-parallel to the strand, thereby forming the core barrel structure.…”

“…For the PKR assay, 17) the reaction mixture contained 54 mM of cinnamoyl-CoA (or cinnamoyldiketide-NAC), 108 mM of malonyl-CoA, 1 mM NADPH, and 5 mg of plant polyketide synthases (A. arborescence pentaketide chromone synthase, 18) octaketide synthase, 19) or Scutellaria baicalensis CHS 17) ), and 60 mg of A. arborescens AKR4C12 (or Glycyrrhiza echinata PKR) in a final volume of 500 ml of 100 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA. Incubations were carried out at 30°C for 20 min to overnight, and the products were analyzed by the reversephase HPLC as described before.…”

“…[4][5][6][7] Although the buried nature of the CHS active-site precludes PKR from accessing the CHS-enzyme-bound polyketide intermediate, PKR is thought to reduce a carbonyl group of a linear polyketide intermediate (or a non-aromatized 1,3,5-cyclohexatrione intermediate) prior to formation of the aromatic ring system. 16,17) To further study the plant PKR enzymes, we carried out cloning of PKRs from a medicinal plant Aloe arborescens (Liliaceae) that produces considerable amount of the "reduced" polyketides including anthrones and anthraquinones. 18,19) Here we report a PKR homolog obtained by a combination of RT-PCR using degenerate primers based on the conserved sequences of plant PKRs (chalcone reductases) and cDNA library screening by oligonucleotide hybridization.…”

Structural Basis for the Formation of Acylalkylpyrones from Two β-Ketoacyl Units by the Fungal Type III Polyketide Synthase CsyB

“…Acyl diketideNACs with various lengths were synthesized according to the published method (19,20). Oligonucleotides were obtained from Eurofins Genomics.…”